Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation

Abstract

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast

“…Intermediates transport and metabolon formation are likely involved to facilitate the MIA biosynthesis that is largely located in the cytosol, the lack of which may be a reason for the low yields in our yeast feeding experiments as the intermediates are rapidly decomposed. Recently, several pathogenesis-related 10 proteins (PR10) were found to bind and stabilize intermediates in the benzylisoquinoline alkaloids biosynthesis in opium poppy ( Ozber et al, 2022 ). If similar auxiliary proteins could be identified in C. roseus , their co-expression in yeast might lead to improved intermediate trafficking and overall flux.…”

Strong Feedback Inhibition of Key Enzymes in the Morphine Biosynthetic Pathway from Opium Poppy Detectable in Engineered Yeast

“…Accurate determination of kinetic parameters for SalAT/THS2 was impacted by the unique characteristics of the coupled reaction. THS2 is part of a family of BIA binding proteins abundant in opium poppy latex and involved in alkaloid accumulation. , Although the binding of thebaine to THS2 was shown in a crystal structure, the exact nature of the binding was not clear. In addition to its alkaloid-binding properties, the enzymatic substrate, salutaridinol 7- O -acetate, also acts as an allosteric regulator of THS2 .…”

“…Owing to the labile nature of the SalAT reaction product, salutaridinol-7-O-acetate, and spontaneously converted derivatives, the in vitro assay used to test for enzyme inhibition required the coupling of SalAT and THS2 to monitor thebaine formation accurately. Since THS2 is also an effective BIA binding protein, 52 the ratio of SalAT and THS2 was first optimized to find the lowest effective dose of THS2 at relatively low (3 μM) and high (50 μM) concentrations of the substrate salutaridinol to ensure that THS2 was not rate-limiting. At both tested concentrations of salutaridinol, a 1:2 ratio of SalAT to THS2 was sufficient to facilitate the optimal formation of thebaine (Figure S6).…”

“…Recombinant Enzyme Purification. Construction of the GfNMT1 expression vector 75 and the pACE1-THS2 plasmid 52 was reported previously. The pACE1−4′OMT and pACE1-6OMT vectors were constructed by amplifying 4′OMT and 6OMT from opium poppy (Bea's choice cultivar) stem cDNA with primers containing the sequence coding of N-terminal His-tag and BamHI and SacI restriction sites.…”

California poppy (Eschscholzia californica), the Papaveraceae golden girl model organism for evodevo and specialized metabolism

“…The biosynthesis of morphinan alkaloids in opium poppy requires celltype-specific localization of the biosynthetic enzymes. In situ localization of their transcripts indicated that seven biosynthetic enzymes (6OMT, CNMT, CYP80B, 4'OMT and BBE involved in reticuline biosynthesis, and SAT and COR in the morphine pathway) were localized in sieve elements, whereas proteins were localized in the supporting companion cells, demonstrating a complex spatial organization of morphinan alkaloids (Lee et al, 2013;Ozber et al, 2022). California poppy could serve as preferred host system to reconstruct the morphinan pathway including genes encoding for the biosynthetic enzymes, translocators, and transcription factors genes involved in cell differentiation.…”